Structural changes in cytochrome p-450(cam) effected by the binding of the enantiomers (1R)-Camphor and (1S)-Camphor

A comparative study of the enantiomeric substrate [(1R)-camphor- and (IS)-camphor)-bound cytochrome P-450(cam) concerns the spin-state equilibrium, substrate dissociation, the thermal unfolding of the protein structure, and the subconformer equilibria observed in the infrared spectra of the carbon monoxide (GO) complex of cytochrome P-450(cam). The behavior of the different conformational equilibria in dependence on temperature, pressure, pH-value, cosolvent, and cation binding led us to suggest that (1S)-camphor is more loosely and less optimally bound in the heme pocket, which facilitates the access of solvent molecules into the heme-iron environment, The spin reaction volume difference measured using the high pressure technique is smaller by 16 +/- 9 cm(3)/mol for (1S)-camphor-bound P-450(cam) compared to the (1R)-camphor-bound P-450(cam), which might indicate a higher water content in the protein and in the heme environment in the (IS)-camphor complex, The half-transition temperature of the thermal unfolding of 53.8 degrees C for the (1S)-camphor-bound oxidized cytochrome P-450(cam) is one degree lower than the value for the (IR)-camphor-bound protein (54.8 degrees C), In the reduced, GO-bound form of cytochrome P-450(cam) at 290 K the (1S)-camphor complex reveals another CO stretch vibration population distribution with slightly higher frequencies [1940.2 cm(-1) (major band) and 1946.3 cm(-1) (minor band)] compared to the (1R)-camphor complex [1939.7 cm(-1) (major band) and 1930 cm(-1) (minor band)], A loosening of the contact between the iron-bound CO ligand and amino acids of the I-helix, probably induced by compensating effects of the increased water content, is suggested. Assuming the carbon monoxide complex as a model for the dioxygen complex, the more loosened binding of (1S)-camphor, therefore the increased water accessibility, and the weaker contact of the iron ligand to the I-helix might explain the higher amount of uncoupling of the cytochrome P-450 reaction cycle compared to that when (1R)-camphor is used as substrate.